THE JOURNAL OF BIOLOGICAL CHEMISTRY
8644 –8652, 1997
© 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Nuclease Activity of T7 RNA Polymerase and the Heterogeneity of
Transcription Elongation Complexes*
(Received for publication, October 11, 1996, and in revised form, January 16, 1997)
Srinivas S. Sastry‡ and Barbara M. Ross
From the Laboratory of Molecular Genetics, Box 174, The Rockefeller University, New York, New York 10021
We have discovered that T7 RNA polymerase, purified
to apparent homogeneity from overexpressing Esche-
richia coli cells, possesses a DNase and an RNase activ-
ity. Mutations in the active center of T7 RNA polymerase
abolished or greatly decreased the nuclease activity.
This nuclease activity is specific for single-stranded
DNA and RNA oligonucleotides and does not manifest
on double-stranded DNAs. Under the conditions of pro-
moter-driven transcription on double-stranded DNA, no
nuclease activity was observed. The nuclease attacks
DNA oligonucleotides in mono- or dinucleotide steps.
The nuclease is a 3* to 5* exonuclease leaving a 3*-OH
end, and it degrades DNA oligonucleotides to a mini-
mum size of 3 to 5 nucleotides. It is completely depend-
ent on Mg21. The T7 RNA polymerase-nuclease is inhib-
ited by T7 lysozyme and heparin, although not
completely. In the presence of rNTPs, the nuclease ac-
tivity is suppressed but an unusual 3*-end-initiated po-
lymerase activity is unmasked. RNA from isolated pre-
elongation and elongation complexes arrested by a
psoralen roadblock or naturally paused at the 3*-end of
an oligonucleotide template exhibited evidence of nu-
clease activity. The nuclease activity of T7 RNA polym-
erase is unrelated to pyrophosphorolysis. We propose
that the nuclease of T7 RNA polymerase acts only in
arrested or paused elongation complexes, and that in
combination with the unusual 3*-end polymerizing ac-
tivity, causes heterogeneity in elongation complexes.
Additionally, during normal transcription elongation,
the kinetic balance between nuclease and polymerase is
shifted in favor of polymerase.
Transcription by procaryotic DNA-dependent RNA polym-
erases can be represented as follows (Scheme I).
k1 k2 NTPs mRNA
RNAP 1 P -
| 0 RPc-
| 0 RPo O R-DNA-RNA
k21 k22 nDNA
1 RNAP P whereas other roadblocks such as psoralens (3, 4), acetoamino-
Initiation Elongation Termination
SCHEME I. Representation of a transcription cycle.
* This work was supported in part by a Louis B. Mayer Foundation
grant and a Hewlett-Packard Company Foundation grant for a high
pressure liquid chromatography work station and related service con-
tracts. This work was presented at a FASEB Summer Research Con-
ference, July, 1995. The costs of publication of this article were defrayed
in part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
‡ A Louis B. Mayer Foundation Fellow. To whom correspondence
should be addressed: Laboratory of Molecular Genetics, Box 174, The
Rockefeller University, New York, NY 10021. Tel.: 212-327-8987; Fax:
212-327-8651; E-mail: sastrys@rockvax.rockefeller.edu.
8644
Vol. 272, No. 13, Issue of March 28, pp.
Printed in U.S.A.
RNAP1 is RNA polymerase, P is promoter, RPc is a closed
complex, RPo is an open complex, k is a rate constant for the
various steps, and NTPs are ribonucleoside triphosphates.
Initiation involves binding of RNAP holoenzyme to promoter
DNA (RPc) and the isomerization to open complexes (RPo) (for
a review, see Ref. 1). Open complexes synthesize short RNAs
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(up to 10 nts) during abortive initiation. After clearing the
promoter, RNAP enters the elongation phase. Termination oc-
curs at certain DNA sequences in either a factor-dependent or
a factor-independent manner (2). The current view of transcrip-
tion elongation has been possible because of our ability to
arrest elongation at specific sites on DNA templates, partial
purification of arrested complexes, and the enzymatic and
chemical probing of their structures (3–17).
The unexpected discovery of an RNA cleavage reaction in
arrested Escherichia coli complexes (18) was followed by docu-
mentation of analogous cleavage reactions in other RNAPs
(19 –23). E. coli RNAP and eucaryotic RNAP II are capable of
RNA cleavage in binary and ternary complexes (24 –26). GreA
and GreB of E. coli enhance the intrinsic cleavage by E. coli
RNAP (25, 27, 28). GreA and GreB (and a eucaryotic counter-
part, SII) prevent elongation arrest (29). GreA may participate
in the fidelity of RNA synthesis (30).
T7 RNAP (98.8 kDa) belongs to a class of single-subunit
RNAPs that includes T3 and SP6 phage RNAPs (31–33). The
three-dimensional structure of T7 RNAP shows high a-helicity
with a deep cleft. Using a novel photochemical cross-linking
technique, we have identified the T7 RNAP cleft as the site of
promoter binding (34). The polymerase shows a striking struc-
tural similarity to E. coli DNA polymerase Klenow fragment
and HIV reverse transcriptase (35). In vitro, T7 RNAP tran-
scribes DNA without additional protein factors. Promoter com-
plexes have been characterized by footprinting and low resolu-
tion NMR (36, 37). T7 RNAP has a higher rate of elongation
(;250 nts/s) than E. coli RNAP (40 –50 nts/s). Elongation com-
plexes are remarkably resilient in their elongating phase. T7
RNAP can transcribe past noncovalently bound proteins (38),
DNA triple helices (39), and some base-specific adducts (40),
fluorine (40), and Z-DNA antibodies (41) can temporarily block
elongation. RNA cleavage by bacteriophage T7-like RNAPs has
not been demonstrated so far. Part of the problem has been the
difficulty of isolating partially purified T7 RNAP elongation
complexes in a stable register.
Here, we show that T7 RNAP also exhibits a novel DNA and
RNA cleaving property. RNase activity occurs in ternary elon-
gation complexes and in binary complexes. In combination with
1
The abbreviations used are: RNAP, RNA polymerase; T7 RNAP,
bacteriophage T7 RNA polymerase; nt(s), nucleotide(s); XL, interstrand
psoralen-DNA cross-link; TBE, 180 mM Tris borate, pH 7.5, 2 mM EDTA
buffer; HIV, human immunodeficiency virus; ss, single-stranded; ds,
double-stranded; MC, multimeric complex; pol II, polymerase II; GpG,
guanylyl-39-59-guanosine phosphate; NMP, nucleoside monophosphate.
This paper is available on line at http://www-jbc.stanford.edu/jbc/
 Nuclease Activity Associated with T7 RNA Polymerase
an unusual 39-end-initiated polymerase activity, the RNase
activity produces heterogeneity in stalled/arrested T7 RNAP
elongation complexes.
EXPERIMENTAL PROCEDURES
Nucleic Acids and Proteins—DNA and RNA oligonucleotides were
commercially synthesized by automated solid phase procedures. DNAs
were purchased from Midland Certified Reagent Co. (Midland, TX), and
RNAs were purchased from the Rockefeller University biotechnology
facility. The plasmids pBluescript and pBR322 were prepared using
standard protocols (42). The concentrations of nucleic acids were calcu-
lated from their respective molar extinction coefficients at 260 nm
(;104 M21zcm21/nt). rNTPs and dNTPs were purchased from Pharmacia
Biotech Inc. at a concentration of 100 mM. T4 polynucleotide kinase and
calf terminal transferase were from New England Biolabs and Boer-
hinger Mannheim, respectively. Heparin sulfate was from Sigma. T7
lysozyme and anti-T7 RNAP antiserum were a kind gift from Dr. F. W.
Studier (Brookhaven National Laboratory, Upton, NY).
Nuclease Assays—T7 RNAP was prepared locally according to the
procedure of Grodberg and Dunn (43) and also by a procedure that was
modified from Ref. 44. We also used preparations of T7 RNAP that were
prepared by a procedure from the Uhlenbeck laboratory (45). His-
tagged T7 RNAP was overexpressed from a plasmid (pBH161) in E. coli
cells (BL21). The enzyme was purified using a Ni21-agarose column.
The bacterial strains overexpressing the His-tagged T7 RNAP, the
strains containing the insertion mutations in the T7 RNAP structural
gene, and the procedure for purification of His-tagged T7 RNAP were a
generous gift from Dr. Bill McAllister (State University of New York at
Brooklyn). Other sources of T7 RNAP are mentioned under “Results.”
The concentration of the purified enzyme was determined using e280 5
1.4 6 0.1 3 105 (43). For nuclease assays, 2 pmol of 59- or 39-end-labeled
DNAs or RNAs were mixed with 5–10 pmol of T7 RNAP in a 25-ml
reaction containing transcription buffer (50 mM Tris-HCl, pH 8.0, 10
mM MgCl2, 1 mM dithiothreitol, 1 mM spermidine, 5% (v/v) glycerol). The
reaction mixture was incubated at 37 °C for various lengths of time
during which 2- or 4-ml aliquots of the reaction were taken out at
different times and mixed with 4 or 10 ml of 8 M urea-Tris borate, 20 mM
EDTA plus tracking dyes and heated in a boiling water bath for 5 min
and run on a 24% acrylamide, 8 M urea gel (19:1 acrylamide:bisacryl-
amide). The cleavage reaction was visualized by autoradiography with
an x-ray film or phosphor screen. Where necessary, the kinetics of the
cleavage reaction was measured by quantitation of the residual full-
length 23-mer DNA band or 20-mer RNA band at specific intervals
relative to the band representing the starting material, i.e. the 0-min
time point. Semiquantitative data were obtained using the ImageQuant
program of a PhosphorImager.
Preparation of 66-mer DNA Templates—Two templates were pre-
pared, one unmodified and the other with a psoralen cross-link (be-
tween T at the 136 position on the top strand and T at the 137 position
of the bottom strand (66XL); see Fig. 7). The 66XL template was
prepared as described previously (Ref. 3; see Ref. 46 for methods of
adduct preparation). Briefly, a 14-mer furanside monoadduct
(CGAAGCTACGAGCA) was ligated to a synthetic, kinased 52-mer (59-
GAGGCCATCGATAAAGGTCTAGATCTCCCTATAGTGAGTCGTATT-
AATTAGC-39) and a 13-mer (59-GGCCTCTGCTCGT-39). The 13-mer
DNA served as a ligation “bridge” between the 14-mer and the 52-mer.
An equimolar amount of a synthetic 66-mer nontemplate strand was
then added and the 66-mer furanside monoadduct bottom strand (the
ligation product) was cross-linked to the nontemplate strand with 320 –
380 nm light. The 66XL was then purified from preparative denaturing
gels.
Analysis of Elongation Complexes—Unmodified 66-mer ds template
was constructed from two complementary strands that were synthe-
sized individually and purified by high performance liquid chromatog-
raphy. The sequence of the template is the same as the one previously
used (see Fig. 7 for sequence) (3, 4). To construct an unmodified 66-mer
template equimolar amounts of a mixture of the two complementary
strands (1 mM each) were heated at 70 °C for 10 min and slowly cooled
to room temperature over a period of 2 h. When needed, the template
strand was 32P-labeled with the aid of [g-32P]ATP and T4 polynucleotide
kinase (42). Psoralen cross-linked templates were constructed as de-
scribed above. The psoralen site specifically cross-linked the T at the
136 position on the top strand with the T at the 137 position on the
bottom strand (see Fig. 7). Typical transcription reactions were carried
out by first mixing ;80 pmol of template DNA (either 32P-labeled at the
59-end of the template strand or unlabeled) in transcription buffer
containing 1 mM each of cold ATP, GTP, and UTP, cold CTP at 50 mM,
8645
FIG. 1. Nuclease activity of T7 RNAP on a 5*-end-labeled ss
Downloaded from www.jbc.org at SEOUL NATL UNIV COLL OF MED, on April 5, 2010
DNA and ss RNA. The autoradiogram of a 24% acrylamide gel shows
the cleavage patterns of DNA and RNA at different intervals of time.
Aliquots were removed at indicated time intervals from reaction mix-
tures containing purified T7 RNAP plus a 23-mer DNA or a 20-mer
RNA. Lane 1 shows the oligonucleotides before polymerase addition,
whereas lane 2 (0.5 min), lane 3 (1 min), lane 4 (5 min), lane 5 (10 min),
lane 6 (20 min), lane 7 (30 min), and lane 8 (60 min) show the oligonu-
cleotides after polymerase addition. WT is wild type polymerase and
MUT is mutant polymerase containing an insertion (pLG12; see Ref.
47). The two less intense shorter bands aside from the full-length DNA
oligonucleotide band in lane 1 are failure sequences during solid phase
synthesis. The presence of these minor bands does not compromise the
interpretation of our data.
[a-32P]CTP (specific activity, 3000 Ci/mmol) at 0.3 mM, and 60 units of
human placental RNase inhibitor. Transcription was initiated by the
addition of 400 pmol of T7 RNAP and incubation at 37 °C. After 15 min
of transcription, 300 mM NaCl or 90 mg/ml heparin was added. When
necessary, sodium pyrophosphate (Sigma) was added at a final concen-
tration of 2 mM after the addition of NaCl. After 3 min at room tem-
perature, the entire reaction was passed through a Sephadex G-25 spin
column. The flow-through was concentrated by SpeedVac centrifuga-
tion at room temperature (;10 min) to about 50 –100 ml, and glycerol
was added to 5% final concentration. The reaction was loaded on an 8%
acrylamide nondenaturing gel (14 cm 3 16 cm) and run at 10 V/cm. For
some reactions, the spin column step was eliminated to achieve a better
yield of the complexes. The gel was run until the blank xylene cyanol
dye was at the bottom of the gel. C1 and C2 bands were identified by
autoradiography of the wet gel, and the respective complexes were
eluted from gel pieces by electroelution. The nucleic acids were recov-
ered by precipitation with EtOH and run on a 24% acrylamide, 8 M
urea-TBE gel. The sizes of the RNAs were measured using markers
generated by diethyl pyrocarbonate plus piperidine (purines) cleavage
of DNA oligonucleotides whose sequences are known. Additional mark-
ers were a synthetic DNA ladder ranging from 8 to 75 nts. RNA
migrated about 2–2.5 nts slower than DNA of the same length on 24%
denaturing acrylamide gels.
RESULTS
Single-stranded DNA Cleavage by T7 RNAP—Fig. 1 shows
that purified T7 RNAP from overexpressing E. coli cells cleaved
a 59-32P end-labeled 23-mer ss DNA (59-TAATACGACTCAC-
TATAGGGAAG-39, promoter top strand) and a 20-mer RNA
(59-UUUUUUUUUUCUGACUUAGC-39). The cleavage oc-
curred from the 39-end (see below) in steps of mono- and
dinucleotides (Fig. 1, lanes 2– 8). After 60 min of DNA diges-
tion, major products of 2 or 3 nts were seen. The pattern of DNA
and RNA fragments was the same, except that the DNA oligo-
nucleotide was cleaved to smaller products faster than the RNA
oligonucleotide. The 20-mer RNA migrates about 2–2.5 nts
slower compared with the same-sized DNA. Hence, in Fig. 1,
the RNA and DNA bands appear to migrate approximately at
the same positions on the high resolution gel (24% acrylamide).
To rule out the possibility that the nuclease activity was due to
an adventitious contaminant in the enzyme preparations, we
8646 Nuclease Activity Associated with T7 RNA Polymerase

FIG. 2. In situ assay for nuclease activity. A, 10% acrylamide-SDS gel showing an example of T7 RNAP preparation used in this work. The
protein was stained with Coomassie Brilliant Blue. B, native (without SDS) 10% acrylamide gel stained with Coomassie Brilliant Blue. MC is a
multimeric complex of T7 RNAP and M is the monomer. S contains native gel standards from Pharmacia. The standards are: 669 kDa,
thyroglobulin; 440 kDa, ferritin; 232 kDa, catalase; 140 kDa, lactate dehydrogenase; 67 kDa, albumin. C, binding of 32P-labeled 23-mer to
multimeric complex of T7 RNAP. After electrophoresis at 4 °C, the native gel was rinsed at 4 °C by agitating in three changes of transcription buffer
for 3 h. The final rinse was discarded, and the gel was shaken overnight at room temperature in 30 ml of transcription buffer containing 32P-labeled
23-mer (1 pmol/ml). The gel was then rinsed three times (15 min each) in transcription buffer and autoradiographed. The MC band was identified
by aligning the autoradiogram with the gel after staining with Coomassie Brilliant Blue (as in B). D, denaturing gel showing the isolated DNA from
C. Lane 1 contains an aliquot of the 32P-labeled 23-mer DNA from the binding solution used in C. Lane 2 contains DNA eluted from band MC from

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the gel in C. Lane S contains synthetic DNA oligonucleotide standards.

purified mutant T7 RNAPs containing insertion and point mu-
tations in the structural gene of T7 RNAP. These mutants were
cloned in Dr. McAllister’s laboratory (see Refs. 47 and 48 for
details). We prepared T7 RNAPs from four mutant strains
containing plasmids pLG12, pLLG22, pWJC22, and pCAR27.
All four mutant T7 RNAPs were defective in DNA and RNA
cleavage to different extents. In Fig. 1 we show an example of
a cleavage pattern that was observed with the pLG12 T7 RNAP
insertion mutant (MUT). This mutant had a 6-base pair linker
insertion within or immediately after codon 566 in bacterio-
phage T7 gene 1 (the structural gene for T7 RNAP) (47). This
mutant was defective in promoter binding and catalysis (47). In
our assay, the mutant enzyme was defective in both DNA and
RNA cleavage (Fig. 1). Surprisingly, the mutant T7 RNAP
behaved somewhat differently with DNA and RNA oligonucleo-
tides. Whereas DNA cleavage occurred to some extent, albeit at
a slower rate than with wild type T7 RNAP, RNA cleavage was
almost completely absent (Fig. 1). For example, with the mu-
tant T7 RNAP, after 60 min of incubation with ss DNA, bands
extending up to 17-mer were visible, the most intense bands
being the 11-mer and 12-mer (Fig. 1, MUT, lane 8). Whereas
with wild type T7 RNAP, at 60 min a much smaller distribution
was observed (Fig. 1, WT, lane 8). This experiment showed that
1) the nuclease activity is indeed due to the polymerase itself;
2) because the overall pattern of cleavage of both ss RNA and ss
DNA appears to be the same, a similar mechanism of cleavage
probably occurred; 3) the cleavage rates are different for ss
DNA versus ss RNA for both wild type and mutant, perhaps
due to differential affinities of the polymerase for ss DNA
versus ss RNA (indeed, using gel-shift assays, we found that T7
RNAP binds ss DNA better than ss RNA; not shown); and 4)
because the mutant T7 RNAP is defective in promoter binding
and catalysis (47) and is also defective in our cleavage assay
here, cleavage is probably catalyzed by the active center of the
enzyme.
Four other T7 RNAP preparations were also tested for nu-
clease (data not shown): T7 RNAP that was prepared using the
procedure described in Ref. 45; His-tagged T7 RNAP that was
prepared using a Ni21-agarose column (bacterial strains and
procedure from Dr. W. T. McAllister); T7 RNAP that was pur-
chased from Epicenter Technologies (Madison, WI) (43) and
modified by Dr. J. J. Jendrisak (Epicenter Technologies); and
T7 RNAP from Dr. J. J. Dunn (Brookhaven National Labora-
tory, Upton, NY) (43). All these purified preparations displayed
the same patterns of cleavage as in Fig. 1. Cleavage activity
also occurred on a 66-mer ss DNA or a 66-mer ds DNA with a
39 nucleotide overhang (not shown). All these oligonucleotides
contained a T7 RNAP promoter sequence. Nuclease assays
using a 59 32P-end-labeled 34-mer ss DNA without promoter
sequence (59-CGAAGCTACGAGCGGTAGCCATCGATAAAT-
AGCT-39) gave the same result (not shown). Therefore, pro-
moter sequence is not required for T7 RNAP nuclease activity.
To further rule out the possibility of contaminating nuclease
in our T7 RNAP preparations we carried out the following
experiments. SDS-polyacrylamide gel electrophoresis showed
the presence of a single band migrating at ;99 kDa after
staining with Coomassie Brilliant Blue (Fig. 2A) or with silver.
We estimated that these preparations were .90 –95% pure T7
RNAP. When T7 RNAP was run on a native (i.e. without SDS)
polyacrylamide gel only two distinct bands were seen after
Coomassie Brilliant Blue staining (Fig. 2B). A multimeric com-
plex (MC) was observed. Since T7 RNAP is known to form
aggregates in low salt,2 it is possible that a large fraction of T7
RNAP migrates close to a 669-kDa marker apparently as a
multimer in native gels containing low concentrations of salt.
However, it is difficult to assign a mass to this multimer based
solely on native gels. Hence, we shall simply refer to this as a
MC. A second, fainter band, apparently corresponding to a
monomer (Fig. 2B, M), based on its electrophoretic mobility,
was also observed. Because this was a native gel (i.e. without
SDS), we are not certain whether band M represented the
uncleaved ;99-kDa T7 RNAP molecule or the “nicked” 80-kDa
fragment that is often seen in polymerase preparations (43).
There was no visible amount of the 80-kDa fragment in our T7
RNAP in SDS gels (Fig. 2A). This is because we prepared T7
RNAPs from E. coli strain BL21 (without Omp T protease; see
Ref. 43). Since the same T7 RNAP preparation was run on SDS
and native gels, and because the ;20 kDa (;99 – 80 kDa) was
not seen on native gels, we believe that the lower band probably
represents a monomer of T7 RNAP. However, for the interpre-
tation of the data below, whether band M represents the full-
length monomer or the nicked polymerase is not relevant be-
cause the material in band M did not bind DNA.
To assay for DNA binding we soaked a gel identical to that in
Fig. 2B in nuclease assay buffer containing 59 32P-end-labeled
23-mer DNA. Only the MC band showed a prominent 32P signal
(Fig. 2C). Overexposed autoradiograms (not shown) of the gel
did not reveal DNA binding to band M. The band representing
MC was excised from the gel, and the DNA was eluted and
subsequently run on a denaturing 24% acrylamide, 8 M urea gel
(Fig. 2D, lane 2). It is clear that cleavage of the 23-mer DNA
2
J. J. Dunn, personal communication.
 Nuclease Activity Associated with T7 RNA Polymerase
FIG. 3. Gel-shift assay for DNA binding to T7 RNAP. 32P-Labeled
23-mer DNA was mixed with T7 RNAP at different molar ratios of T7
RNAP:DNA in transcription buffer. The reaction mixture was incu-
bated at 37 °C for 10 min and made 5% glycerol. The reaction was
loaded on an 8% acrylamide, TBE gel (20 cm 3 20 cm) and run at room
temperature (7 V/cm). The gel was run until the bromphenol blue dye
was 9 cm from the bottom of the gel and then fixed in 5% methanol, 5%
acetic acid, 3% glycerol for 20 min, dried, and autoradiographed. 0
indicates no polymerase. 1, 3, 5, and 15 indicate -fold excess of T7 RNAP
over DNA.
occurred in the T7 RNAP complex. The in situ cleavage pattern
is qualitatively similar to the cleavage pattern in solution (com-
pare Fig. 1, WT, lanes 2 and 3, with Fig. 2D, lane 2). These
experiments show that T7 RNAP may exist as a multimer that
cleaves 32P ss DNA. Next, we did a corollary experiment to
demonstrate that T7 RNAP indeed binds to the ss DNA oligo-
nucleotide in stoichiometric amounts. Fig. 3 is a gel-shift assay
demonstrating that increasing amounts of T7 RNAP bind to the
23-nt ss DNA. A single major upper band (U) and two faster
migrating minor bands (L) are seen (Fig. 3). The faster migrat-
ing shifted bands may represent the 59 cleavage products that
are still bound to the enzyme, consistent with the results from
the in situ cleavage experiment. Alternatively, they may be
faster migrating conformational isomers of the enzyme-ss DNA
complexes. These experiments demonstrate that T7 RNAP
binds and cleaves ss DNA in a stepwise manner reminiscent of
a 39 to 59 exonuclease. Gel-shift assays with 59 32P-end-labeled
20-mer RNA also showed that T7 RNAP was bound to RNA in
binary complexes (not shown).
Additional criteria were used to show that the cleavage ac-
tivity is a property of T7 RNAP. 1) The kinetics of heat inacti-
vation of T7 RNAP at 70 °C indicated that the destruction of
nuclease activity paralleled the destruction of transcription
activity (not shown). 2) Amino acid composition analyses of two
of our preparations matched the known amino acid composition
of T7 RNAP. 3) Western blotting of gel shifts of binary com-
plexes of T7 RNAP and 23-nt DNA (such as those in Fig. 3) and
transcription complexes (see Fig. 8) with anti-T7 RNAP anti-
body indicated that these complexes contained T7 RNAP. 4)
DNA cleavage activity during T7 RNAP purification coincided
with T7 RNAP peak fractions from the cation-exchange
column.
ss DNA Cleavage Activity Starts at the 39-End and Is Mg21-
dependent—When the 23-mer ss DNA was 32P-labeled at the
39-end, the nuclease immediately released a mononucleotide
(Fig. 4). This was deduced using 32P-labeled dinucleotide ([a-
32
P]GpG) and 32P-labeled ATP as markers on a 24% acrylamide
denaturing gel. The product size remained the same even after
prolonged incubation. This indicated that the initial product of
the cleavage is probably a mononucleotide. (Within the scope of
the present paper we do not think it is necessary to unambig-
uously identify this product.) The same result was seen with 39
32
P-end-labeled 34-mer nonpromoter ss oligonucleotide DNA
(not shown). Putting together the 59- and 39-end labeling ex-
periments, T7 RNAP nuclease acts by a stepwise removal of
nucleotides starting from the 39-end and going toward the
8647
FIG. 4. Cleavage of 3*-end-labeled 23-mer DNA by T7 RNAP.
The 23-mer DNA was labeled at its 39-end with [a-32P]ddATP (11) and
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terminal deoxynucleotidyl transferase. Cleavage reactions were carried
out as described under “Experimental Procedures.” 0 indicates no po-
lymerase. 0.5, 2, 5, and 10 indicate min after addition of polymerase.
59-end with the limit product at the 59-end being 3–5 nucleo-
tides. That cleavage left a 39-OH end was verified by 32P label-
ing the cleaved unlabeled 3-mer and 20-mer with 39 terminal
transferase and [a-32P]ddATP after the nuclease assay. Omis-
sion of MgCl2 or inclusion of molar equivalents of EDTA with
Mg21 blocked cleavage, indicating complete Mg21 dependence
of the nuclease (not shown).
Nuclease Activity Is Inhibited by T7 Lysozyme or Heparin—A
203 excess (w/w) of T7 lysozyme over T7 RNAP or heparin (250
mg/ml) inhibited the rate of nuclease activity (Fig. 5). After
5–30 min of incubation with T7 lysozyme, 21-mer and 20-mer
or 10 –12-mers were still seen, whereas without lysozyme the
DNA was cleaved faster to much shorter products. Prolonged
incubation with these reagents showed smaller products, indi-
cating that these reagents did not completely block nuclease
activity. The inhibition by T7 lysozyme or heparin was
concentration-dependent (not shown). Half-maximal inhibition
by heparin was at 50 mg/ml. T7 lysozyme is a specific inhibitor
of T7 RNAP (49, 50), again indicating that T7 RNAP is the
source of nuclease activity. Because the polyanion heparin is a
competitive inhibitor of DNA binding, DNA binding is a pre-
requisite for T7 RNAP nuclease activity.
Polymerase Activity Suppresses Nuclease Activity—Addition
of rNTPs suppresses the nuclease activity while unmasking an
unusual polymerase activity (compare lanes without NTPs and
with rNTPs, Fig. 6). The 39-end extension of DNA (or RNA, not
shown) by T7 RNAP with rNTPs has been observed previously
(51–53). Prolonged incubation in the presence of rNTPs shows
evidence of nuclease activity on the 40-mer DNA-RNA chime-
ras (e.g. see 30-min lane of Fig. 6). Consistent with T7 RNAP
being the nuclease, dNTPs do not elicit the same effect as
rNTPs (Fig. 6). This showed that the active site of T7 RNAP is
involved in nuclease activity, consistent with our earlier results
with mutant T7 RNAPs (Fig. 1). Fig. 6 shows that nuclease
activity and polymerase activity can manifest independently.
In the presence of NTPs, polymerase activity predominates
over nuclease activity (see below).
Nuclease Activity Is Absent during Normal Transcription—
The plasmid pBluescript (Stratagene Corp., San Diego, CA)
contains a class III promoter but not a T7 terminator. In the
presence of T7 RNAP and rNTPs, a large amount of RNA was
produced by multiple rounds of transcription, indicating that
our T7 RNAP was transcriptionally active. No small cleavage
products originating from the DNA were seen. In the absence of
NTPs, no cleavage of the plasmid was seen either (data not
8648 Nuclease Activity Associated with T7 RNA Polymerase
FIG. 5. Inhibition of cleavage activity by T7 lysozyme and hep-
arin. T7 lysozyme or heparin were mixed with labeled 23-mer DNA in
transcription buffer. To this mixture, kept at 37 °C, T7 RNAP was
added, and aliquots were removed at different intervals of time and run
on 24% acrylamide-urea denaturing gel. 0 indicates no polymerase. 5,
15, and 30 indicate min after addition of polymerase. None indicates
reactions that contained neither lysozyme nor heparin.
shown). To confirm that no cleavage of the plasmid occurred,
we isolated the DNA from gel bands from agarose gel and
restricted the DNA with HindIII and PstI separately. Re-ex-
amining the restricted DNA separately on agarose and dena-
turing polyacrylamide gels revealed no indication that T7
RNAP cleaved the plasmid with or without NTPs. We observed
similar results with the plasmid pBR322 that has no consensus
T7 promoter, although in this case very little RNA was made.
We examined transcription initiation on a short oligonucleotide
template containing a 23-base pair core promoter sequence
(59-TAATACGACTCACTATAGGGAAG-39). In the presence of
GTP as the sole NTP, T7 RNAP produces a G-ladder that
extends to ;14 nts and then tapers off (71). The G-ladder is
thought to be produced by multiple rounds of slippage synthe-
sis at the three cytidines in the promoter (54). The G-ladder
accumulated in a time-dependent manner, indicating that the
ds template was not destroyed and the RNA was not degraded
(71). We also examined transcription on an unmodified 66-mer
DNA template (71). During abortive transcription, there was a
time-dependent accumulation of transcripts but no evidence of
template cleavage (71). These observations indicate that in the
presence of NTPs during normal transcription no T7 RNAP
DNase activity is seen.
Isolation of Elongation Complexes—Next, we examined RNA
cleavage activity in isolated ternary elongation complexes. Be-
cause T7 RNAP elongation complexes are relatively unstable
(dwell times of ;5–15 min) we were not able to “walk” discrete
and isolated T7 RNAP elongation complexes in a manner that
was done with E. coli and eucaryotic RNAP elongation com-
plexes. To circumvent these problems, an alternative protocol
(albeit a less “clean” strategy compared with traditional walk-
ing methods) to examine the RNA component in elongation
complexes was developed. Transcription was initiated by the
addition of T7 RNAP to a synthetic oligonucleotide template in
the presence of NTPs. Following 15 min of transcription, NaCl
(to 300 mM) or heparin was added. Addition of high salt or
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FIG. 6. Polymerase activity suppresses nuclease activity in the
presence of rNTPs. Reaction mixtures contained either rNTPs or
dNTPs before the addition of T7 RNAP. 0 indicates no polymerase. 1, 5,
and 30 indicate min after addition of polymerase.
heparin disrupted all existing promoter complexes and inhib-
ited new rounds of initiation. Footprinting and other experi-
ments have shown that 300 mM NaCl disrupted promoter-
initiation complexes but not elongation complexes (3, 4, 55–57).
To isolate stable complexes, we first passed the transcription
reactions through a Sephadex G-25 spin column to remove
NTPs and other low molecular weight components. The radio-
labeled complexes were resolved on a low percentage nondena-
turing acrylamide gel. The 32P label was present either in the
nascent RNA or on the 59-end of the template strand. To ensure
that the resolved complexes seen on nondenaturing gels con-
tained T7 RNAP, we probed the complexes with anti-T7 RNAP
antiserum using Western blotting techniques (not shown). The
same bands that were visible in autoradiograms when the RNA
or the DNA templates were labeled with 32P were also recog-
nized by anti-T7 RNAP antibody, attesting that the complexes
indeed contained T7 RNAP. Omission of the spin column step
increased the yield of the complexes without changing the
pattern of bands on the nondenaturing gel. Following autora-
diography of the native gels, the radioactive RNAs were recov-
ered and subsequently resolved on a 24% denaturing acrylam-
ide gel (see “Experimental Procedures”). This protocol selected
high salt or heparin-resistant stable elongation complexes.
We examined the RNA composition in two different T7
RNAP ternary complexes: 1) elongation complexes apparently
paused at the end of an unmodified 66-mer ds DNA template;
and 2) elongation complexes arrested by a psoralen cross-link
site specifically placed toward the 39-end of the same 66-mer
(66XL) template (3).
Cleavage Reactions in End-paused Elongation Complexes—
Fig. 7 shows the sequence of the 66-mer DNA for examining
end-paused and psoralen-arrested complexes. Footprinting
demonstrated that elongating T7 RNAP, as indicated by the
box in Fig. 7, was blocked by the psoralen cross-link (3). Here,
we made the unmodified as well as the psoralen cross-linked
template to study cleavage and end-polymerization reactions.
On the unmodified 66-mer template, the elongation complexes
paused at the end of the template (end-paused complexes). The
position of the polymerase molecule in end-paused complexes is
suggested by the box in Fig. 7. Although we have not foot-
printed the end-paused elongation complexes, we assume that
these complexes occupied a similar overall position at the end
of the template as the psoralen-arrested complex (Fig. 7). This
assumption is justified because the majority of the RNA in the
 Nuclease Activity Associated with T7 RNA Polymerase
FIG. 7. Sequence of the 66-mer template used for the analysis of elongation complexes. The box represents the polymerase molecule.
The arrows show the DNase I footprints (3).
isolated complexes is a runoff transcript (43 nts; Fig. 8C).
Using the protocol described in the previous section, we
examined the 32P-labeled RNA in end-paused and psoralen-
blocked complexes. Fig. 8A (with unmodified template) and 8B
(with cross-linked template) show complex 1 and complex 2
with different mobilities. Identical bands were seen when only
the DNA was labeled (not shown). From the specific radioac-
tivity of [32P]RNA or [32P]DNA in gel bands seen in the absence
of high salt, we estimated that complexes contained approxi-
mately an equimolar ratio of DNA and RNA. Two bands were
seen after addition of 300 mM NaCl, which was added after 15
min of transcription. Only elongation complexes were stable
during gel electrophoresis, and they have different mobilities
on nondenaturing gels. When heparin was added, only complex
2 was visible. This is in agreement with previous observations
that heparin caused disruption of some elongation complexes
(4, 58). The ratio of [32P]CMP-labeled C1 to C2 with 66XL was
almost equal (Fig. 8B), whereas with unmodified 66-mer ds
DNA template, C1 accounted for .90% of the total radioactiv-
ity (Fig. 8A). Clearly, the presence of the psoralen cross-link
influenced the relative populations of C1 and C2. The basis for
this difference is unknown.
Examination of 32P-labeled RNA in NaCl- (C1) or heparin-
resistant (C2) complexes revealed a heterogeneous pattern of
transcripts (Fig. 8C). In C1, the major cleavage products were
of 12, 17, 20, 26, and 31 nts, etc., and were interspersed with
minor products with 1- or 2-nt differences. Longer than 11-
initiated transcripts (up to ;60-mers) were also seen. These
transcripts were not due to end initiation (or internal initia-
tions at sites other than 11) because they were observed even
when [a-32P]GpG was used, which forces T7 RNAP to correctly
initiate at 11 (not shown). The 12-nt transcripts are not abor-
tive transcripts because the latter are usually only up to 10 nts
and are generally more abundant relative to the longer RNAs.
Moreover, just after addition of NaCl or heparin and prior to
passage through the spin column, most of the complexes were
end-paused, as indicated by the abundance of the 43-nt runoff
species (Fig. 8C, lane RF). We believe that the heterogeneity is
due to RNA cleavage and end-polymerization reactions analo-
gous to those seen earlier (see Figs. 1 and 6). Heterogeneity
8649
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cannot be due simply to the trapping of polymerase complexes
in various registers of elongation by the spin column/gel. This
would have produced a more or less stochastic distribution of
RNAs. There are other compelling reasons to eliminate the
latter explanation. 1) Addition of 300 mM NaCl or heparin
precluded any further polymerase binding after 15 min of tran-
scription. 2) Even in heparin-resistant complexes (C2 in Fig.
8C) the cleavage and end-extended RNAs are observed. In fact,
more or less the same pattern (down to 22 nts) is seen with both
NaCl and heparin (because of the lower yield of heparin-resist-
ant complexes, the band intensities were lower). 3) Further-
more, examination of the transcription products before spin
column/gel purification (Fig. 8C, lane RF) showed RNAs that
were overwhelmingly in the larger sizes (35– 43 nts), indicating
that just before spin column passage most of the complexes in
solution were close to the end of the template. Because the
NTPs were removed after the passage through the spin column
gel, RNA cleavage and end polymerization must have occurred
prior to the spin column step and/or before native gel electro-
phoresis. The high salt- or heparin-resistant heterogeneous
complexes were trapped by the gel. We believe the heterogene-
ity of transcripts is due to a fraction of the end-paused com-
plexes undergoing end-polymerization and cleavage reactions.
Cleavage Reactions in Psoralen Cross-link-arrested Elonga-
tion Complexes—Next, we examined the RNA composition of
T7 RNAP complexes arrested by an authentic roadblock (pso-
ralen cross-link) (3, 56). Fig. 8D shows that the patterns of
cleavage of nascent RNA transcripts from C1 and C2 complexes
is quite different from the end-paused ones on noncross-linked
template (Fig. 8C). Surprisingly, RNA transcripts were ex-
tremely heterogeneous in both C1 and C2, occurring in a wide
range in 1- or 2-nt steps (11–28 nts; Fig. 8D). The pattern of
cleavage is reminiscent of that in Fig. 1. Transcripts extending
up to ;75 nts were seen in abundance in C2. The 36-mers
expected from transcription up to the psoralen cross-link were
only a minor species. Longer RNAs than those specified by 11
initiation were due to RNA 39-end polymerization, whereas the
shorter RNAs were due to cleavage. Pyrophosphorolysis is the
chemical reversal of nucleotide incorporation by nucleic acid
polymerases. Pyrophosphorolysis is usually accomplished in
8650 Nuclease Activity Associated with T7 RNA Polymerase
FIG. 8. Gel-shift assay for the isolation of T7 RNAP elongation
complexes paused at the end of a template (A) or those arrested
by a psoralen cross-link (B). RNA was labeled with [a-32P]CTP. See
“Experimental Procedures” for details. For a better band resolution, we
ran the free RNA off the gels (2). A, unmodified 66-mer was the
template. B, the template was a psoralen cross-linked 66-mer DNA. C,
high resolution gel analysis of RNAs in end-paused elongation com-
plexes. Lane C1 contains 32P-labeled RNA isolated from NaCl-treated
complexes. Lane C2 contains 32P-labeled RNA isolated from heparin-
treated complexes. RF is an aliquot of labeled RNA from transcription
reactions before spin column gel separation. D, high resolution gel
analysis of RNAs in elongation complexes arrested at a site-specific
psoralen cross-link. Lanes C1 and C2 contain 32P-labeled RNA isolated
from untreated or NaCl-treated complexes.
vitro by millimolar concentrations of PPi (13, 23, 59 – 61). We
did not observe any significant differences in the pattern of
transcripts from psoralen-arrested elongation C1 and C2 com-
plexes following addition of PPi, suggesting that PPi has no
effect on RNA cleavage. In general the yield of complexes was
lower in the presence of NaPPi (data not shown).
DISCUSSION
Nuclease Activity of T7 RNAP—We have discovered that T7
RNAP prepared from overexpressing E. coli cells exhibits
DNase and RNase activity. This is a new enzymatic activity of
T7 RNAP. Many experiments have ruled out the probability of
other contaminating nucleases in our T7 RNAP preparations
(see “Results”). Moreover, T7 RNAP prepared using different
methods and from different sources exhibited nuclease activity.
We cannot completely rule out the possibility that a low mo-
lecular weight protein factor (analogous to GreA) is tightly
bound to T7 RNAP. It is, however, highly unlikely that there
was an adventitious contaminant nuclease unrelated to T7
RNAP. Hydrolytic cleavage of RNAs has been documented with
all tested RNAPs. These include E. coli RNAP, higher eucary-
otic pol II and pol III, vaccinia RNAP, and yeast RNAP (see
Introduction for Refs.). In at least two cases (E. coli and yeast
pol II) the cleavage of RNA has been demonstrated in binary
complexes as well as in ternary complexes. Here, both binary
and ternary complexes of T7 RNAP exhibit nuclease activity.
The DNase activity of T7 RNAP leaves a 39-OH end and a
59-PO4 and is absolutely dependent on Mg21. The nuclease
appears to be a 39 to 59 exonuclease on single-stranded oligo-
nucleotide. Although we have not isolated and analyzed the
first cleavage product to the 39-end, based on the pattern of
cleavage of 59-end- or 39-end-labeled DNA and RNA we can only
surmise that the cleavage occurs in mono- or dinucleotide steps
(Figs. 1 and 4). The 59 cleavage products appear to be bound to
the enzyme as indicated by the results from the in situ cleavage
assay (Fig. 2). Multiple T7 RNAP molecules may be involved in
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the cleavage of each DNA (distributive mechanism) as indi-
cated by the binding stoichiometry (Fig. 3) and by the fact that
only the multimeric form of the T7 RNAP showed binding and
cleavage of oligonucleotide DNA (Fig. 2). Since T7 RNAP nu-
clease cannot act on covalently closed circular plasmid DNA or
linear oligonucleotide ds DNA, it is not an endonuclease. We
could not detect T7 RNAP nuclease activity on linear blunt
ended ds DNA oligonucleotides in the absence of transcription.
The time course of T7 RNAP RNase activity is slower than that
of the DNase. We believe that DNase and RNase activities are
carried out by the same catalytic pocket because the cleavage
ladders from a 59-end-labeled DNA or RNA are of the same
pattern, in both cases polymerase activity competes with and
suppresses nuclease activity, and T7 RNAP mutants are defec-
tive in cleavage. It is possible that the same binding site can
accommodate both ss RNA and ss DNA. This proposal is con-
sistent with the ability of T7 RNAP to strongly bind ss DNA
and ss RNA oligonucleotides (5, 62) (Fig. 3). Recently, one of us
(S. S.) has shown by photochemical cross-linking that the ss
DNA (and also perhaps ss RNA) binding activity lies in the
fingers (or fingers-palm junction) domain (34).
Previous reports suggest that 39-end NMP addition to RNA
was due to the presence of a self-complementary secondary
structure or partial intermolecular complementarity in the
nascent RNAs (51, 53). These authors suggested that T7 RNAP
binds to free RNA in transcription reactions to form binary
complexes in which end polymerization occurred. In our exper-
iments, we have eliminated this possibility by a multistep
procedure. 1) We passed the transcription reaction through a
Sephadex spin column to remove NTPs. 2) We ran the com-
plexes on native polyacrylamide gels. In this step, we have
carefully identified ternary complexes by 32P labeling either
the DNA or RNA and ensuring that the complexes contained T7
RNAP using Western blotting with anti-T7 RNAP antibody.
We also made sure that when the template DNA or RNA were
independently 32P-labeled, the reactions were run on the same
gel and bands with identical mobilities were recovered from the
gels. 3) We recovered the complexes from gel pieces by electro-
elution and then ran the radiolabeled RNAs from disrupted
complexes on high resolution denaturing acrylamide gels.
These procedures assured us that the complexes isolated from
native gels were indeed ternary complexes. Some elongation
complexes are resistant to heparin, whereas others are not (4,
55, 58, 63). Therefore, we believe that the observed cleavage
and end-polymerization reactions occurred in ternary tran-
scription complexes and not simply in binary complexes of free
RNA and DNA in transcription reactions. During elongation,
ternary complexes of T7 RNAP are highly heterogeneous. This
 Nuclease Activity Associated with T7 RNA Polymerase
FIG. 9. Model for events leading to transcription arrest/pause
and subsequent cleavage/polymerization reactions. A–C repre-
sent stages in the transcription cycle. See text for a description.
heterogeneity is clearly seen in isolated elongation complexes
paused/arrested by a psoralen roadblock or at the end of a
template (Fig. 8). End pausing has been observed with eucary-
otic pol II during transcription on blunt-ended templates (64).
Models for Cleavage and Unusual Polymerization during
Elongation and the Heterogeneity of Complexes—Fig. 9A is a
diagrammatic representation of an elongation complex paused
at the end of the template or arrested by a psoralen roadblock.
The RNAP encloses a “bubble” with a 3-base pair (or up to
7-base pair) RNA-DNA hybrid (3, 37, 55, 65, 71). The size of the
bubble and hybrid in elongation complexes of T7 RNAP have
not been directly measured. However, they can be inferred from
published reports (55, 71). A fraction of the paused/arrested
polymerases may “back up” (Fig. 8B). Backup of the polymerase
was probably facilitated by the inhibition of enzyme turnover
by high salt/heparin. Backing up may be associated with con-
formational rearrangements in the catalytic core of RNAP. The
39 nascent RNA product binding site in the arrested/paused
RNAP may be reorganized such that 39-end RNA-DNA contacts
are lost but the 39 distal contacts are maintained (Fig. 8B). A
part of the 39-end is now partially single-stranded and is held in
a new RNAP site. The binding of the RNA in the new RNAP
site may facilitate or effect cleavage (Fig. 8B). Recent work with
E. coli RNAP indicated that RNA product binding site reorien-
tation and conformational changes occur in paused and ar-
rested complexes (67). We visualize a stalled/paused T7 RNAP
as having two activities, viz. 39-end-initiated polymerization
(Refs. 51, 53, and 66, and this work) and 39 to 59 RNase activity
(this work). These are opposing activities that generate heter-
ogeneity in paused/arrested complexes. Although the catalytic
pocket is probably the site of both nuclease and polymerase
activities, in the presence of NTPs the polymerase activity is
kinetically dominant. This explains why addition of NTP to the
ss DNA oligonucleotide suppresses the nuclease (Fig. 6) and
why during normal processive transcription the nuclease activ-
ity is not observed. The nuclease activity is only kinetically
unmasked when the polymerase is paused or arrested by a
roadblock.
8651
Why does cleavage of ss RNA oligonucleotide occur with
higher than a 1:1 molar ratio of enzyme to RNA, whereas
cleavage in ternary elongation complex occurs at an equimolar
ratio? The two events, binding and catalytic cleavage, occur in
that sequence. In paused/arrested ternary elongation com-
plexes, the RNA is held very tightly by the enzyme. The RNA
and RNAP in these complexes are not in equilibrium with free
RNA and free polymerase, unlike in binary complexes, where
they are in equilibrium with free RNA and free RNAP. Hence,
higher concentrations of polymerase are required to drive by
mass action the equilibrium toward binding and cleavage in
binary complexes.
Role of Nuclease Activity—Comparison of the T7 RNAP nu-
clease activity with the known nuclease activity of other
RNAPs or HIV reverse transcriptase reveals many differences
and some similarities. At first glance, the ss DNA-ss RNA
nuclease activity of T7 RNAP appears to be somewhat different
from the known GreB-enhanced intrinsic cleavage activity of E.
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coli RNAP. The GreB-enhanced E. coli RNAP cleaves the nas-
cent RNA in arrested/paused ternary complexes into fragments
larger than T7 RNAP (24, 25). The T7 RNAP nuclease pattern
resembles that of GreA-enhanced E. coli RNAP, eucaryotic pol
II, or vaccinia RNAP in that these enzymes seem to cut in
mono- or dinucleotide steps (23, 61). T7 RNAP cleavage is
reminiscent of SII-mediated “minor” mode cleavage in yeast pol
II binary complexes (26) because cleavage starts at the 39-end
releasing 1–3 nts at a time. It is not known if E. coli RNAP or
the eucaryotic RNAPs have a single-stranded DNase activity.
T7 RNAP nuclease activity resembles the HIV reverse tran-
scriptase nuclease in that arrest of template-specified polym-
erization is needed (72). The dual activities, viz. the 39-end-
initiated nontemplated polymerization and the nuclease
activity, are common to T7, E. coli, and pol II RNAPs and to
HIV reverse transcriptase (73). Other common features are the
complete requirement for a divalent cation and the apparent
independence from pyrophosphorolysis. Although we have not
demonstrated that cleavage is accompanied by a backward
movement of the T7 RNAP from the end of the template or from
a stalled site, the patterns of the cleavage sites (Fig. 8) are
consistent with this possibility. Because of the relative insta-
bility (dwell times of ,5–10 min) and heterogeneity of T7
RNAP elongation complexes, it is technically difficult or per-
haps impossible to walk T7 RNAP from one register to another
in isolated homogeneous complexes as was done with E. coli
RNAP. In the case of E. coli RNAP and eucaryotic pol II, it has
been speculated that hydrolytic nuclease activity may be an
error-correcting mechanism or a way of rescuing paused/ar-
rested complexes. Similar mechanisms may operate during T7
RNAP transcription. What role might the ss DNase activity
play in vivo? During T7 phage DNA replication in vivo, linear
T7 DNA concatemers are formed through base pairing of ter-
minal repeats. This is accompanied by digestion of surplus
single-stranded DNA (68). The mechanisms of these processes
are not clearly understood. We speculate that digestion of sur-
plus single-stranded DNA serves two functions: 1) production
of dNTPs to augment cellular pools that are channeled for
rapid T7 DNA replication; and 2) reduction of the competition
from ss DNA for T7 DNA polymerase activity (69). There are
three known nucleases produced by the T7 phage. 1) Gene 6
exonuclease (59 to 39), which is normally involved in removal of
primers after lagging strand synthesis, in recombination, and
in the destruction of bacterial DNA. This is a ds DNA-specific
exonuclease (70). 2) Gene 3 endonuclease, which is specific to
single-stranded DNA and may be a resolvase involved in ho-
mologous recombination. 3) 39 to 59 exonuclease of T7 DNA
polymerase, which is an error-correcting activity during T7
8652 Nuclease Activity Associated with T7 RNA Polymerase
DNA synthesis. T7 RNAP nuclease, the fourth nuclease activ-
ity, may serve as an additional nuclease in the digestion of the
surplus single-stranded DNA during T7 DNA replication and
maturation. Since T7 RNAP nuclease is a single-stranded 39 to
59 exonuclease and is suppressed during normal transcription
elongation, the double-stranded form of T7 DNA would not be
its target.
Acknowledgments—We thank Prof. F. W. Studier for the kind gifts of
anti-T7 RNAP antisera and T7 lysozyme and Dr. Bill McAllister for
bacterial strains and suggestions. We appreciate the very helpful sug-
gestions from Profs. Mike Chamberlin and Charles Richardson. We also
thank Dr. Stewart Shuman for critical comments on the manuscript
and Prof. Joshua Lederberg for interest in the project.
REFERENCES
1. McClure, W. R. (1985) Annu. Rev. Biochem. 54, 171–204
2. Richardson, J. P. (1993) CRC Crit. Rev. Biochem. Mol. Biol. 28, 1–30
3. Sastry, S. S., and Hearst, J. E. (1991) J. Mol. Biol. 221, 1091–1110
4. Sastry, S. S., and Hearst, J. E. (1991) J. Mol. Biol. 221, 1111–1125
5. Sastry, S. S., Spielmann, H. P, Hoang, Q. S., Phillips, A. M., Sancar, A., and
Hearst, J. E. (1993) Biochemistry 32, 5526 –5538
6. Shi, Y.-B., Gamper, H., Houten, B. V., and Hearst, J. E. (1988) J. Mol. Biol.
199, 277–293
7. Pavco, P. A., and Steege, D. A. (1990) J. Biol. Chem. 265, 9960 –9969
8. Nudler, E., Kashlev, M., Nikiforov, V., and Goldfarb, A. (1995) Cell 81,
351–357
9. Chan, C. L., and Landick, R. (1993) J. Mol. Biol. 233, 25– 42
10. Kainz, M., and Roberts, J. (1992) Science 255, 838 – 841
11. Krummel, B., and Chamberlin, M. J. (1992) J. Mol. Biol. 225, 239 –250
12. Krummel, B., and Chamberlin, M. J. (1992) J. Mol. Biol. 225, 221–237
13. Metzger, W., Schickor, P., and Heumann, H. (1989) EMBO J. 8, 2745–2754
14. Nudler, E., Goldfarb, A., and Kashlev, M. (1994) Science 265, 793–796
15. Zaychikov, E., Dennissova, L., and Heumann, H. (1995) Proc. Natl. Acad. Sci.
U. S. A. 92, 1739 –1743
16. Erie, D. A., Yager, T. D., and von Hippel, P. H. (1992) Annu. Rev. Biophys.
Biomol. Struct. 21, 379 – 415
17. Das, A. (1993) Ann. Rev. Biochem. 62, 893–930
18. Surratt, C., Milan, S. C., and Chamberlin, M. J. (1991) Proc. Natl. Acad. Sci.
U. S. A. 88, 7983–7987
19. Reines, D. (1992) J. Biol. Chem. 267, 3795–3800
20. Izban, M. G., and Luse, D. S. (1992) Genes Dev. 6, 1342–1356
21. Whitehall, S. K., Bardeleben, C., and Kassavetis, G. A. (1994) J. Biol. Chem.
269, 2299 –2306
22. Guo, H., and Price, D. H. (1993) J. Biol. Chem. 268, 18762–18770
23. Hagler, J., and Shuman, S. (1993) J. Biol. Chem. 268, 2166 –2173
24. Altmann, C. R., Solow-Cordero, D. E., and Chamberlin, M. J. (1994) Proc. Natl.
Acad. Sci. U. S. A. 91, 3784 –3788
25. Orlova, M., Newlands, J., Das, A., Goldfarb, A., and Borukhov, S. (1995) Proc.
Natl. Acad. Sci. U. S. A. 92, 4596 – 4600
26. Johnson, T. L., and Chamberlin, M. J. (1995) Cell 77, 217–224
27. Borukhov, S., Sagitov, V., and Goldfarb, A. (1993) Cell 72, 459 – 466
28. Borukhov, S., Polyakov, A., Nikiforov, V., and Goldfarb, A. (1992) Proc. Natl.
Acad. Sci. U. S. A. 89, 8999 – 8902
29. Kassavetis, G. A., and Geiduschek, E. P. (1993) Science 259, 944 –945
30. Erie, D. A., Hajiseyedjavadi, O., Young, M. C., and von Hippel, P. H. (1993)
Science 262, 867– 873
31. Chamberlin, M. J., and Ryan, T. (1982) in The Enzymes (Boyer, P. D., ed) Vol.
15, pp. 87–108, Academic Press, New York
32. McAllister, W. T. (1993) Cell. Mol. Biol. 39, 385–391
33. McAllister, W. T., and Raskin, C. A. (1993) Mol. Microbiol. 10, 1– 6
34. Sastry, S. S. (1996) Biochemistry 35, 13519 –13530
35. Sousa, R., Chung, Y. J., Rose, J. P., and Wang, B. C. (1993) Nature 364,
593–599
36. Rastinejad, F., and Lu, P. (1993) J. Mol. Biol. 232, 105–122
37. Muller, D. K., Martin, C. T., and Coleman, J. E. (1989) Biochemistry 28,
3306 –3313
38. Pavco, P., and Steege, D. A. (1991) Nucleic Acids Res. 19, 4639 – 4646
39. Skoog, J. U., and Maher, L. J. (1993) Nucleic Acids Res. 21, 4055– 4058
40. Chen, Y.-H., and Bogenhagen, D. F. (1993) J. Biol. Chem. 268, 5849 –5855
41. Droge, P., and Pohl, F. (1991) Nucleic Acids Res. 19, 5301–5306
42. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
43. Grodberg, J., and Dunn, J. J. (1988) J. Bacteriol. 170, 1245–1253
44. Zawadzki, V., and Gross, H. J. (1991) Nucleic Acids Res. 19, 1948
45. Mulligan, J. F., and Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51– 62
46. Spielmann, P. H., Sastry, S. S., and Hearst, J. E. (1992) Proc. Natl. Acad. Sci.
U. S. A. 89, 4514 – 4518
47. Gross, L., Chen, W. J., and McAllister, W. T. (1992) J. Mol. Biol. 228, 488 –505
48. Raskin, C., Diaz, G., and McAllister, W. T. (1993) Proc. Natl. Acad. Sci. U. S. A.
90, 3147–3151
Downloaded from www.jbc.org at SEOUL NATL UNIV COLL OF MED, on April 5, 2010
49. Ikeda, R. A., and Bailey, P. A. (1992) J. Biol. Chem. 267, 20153–20158
50. Moffatt, B., and Studier, W. F. (1987) Cell 49, 221–227
51. Cazenave, C., and Uhlenbeck, O. C. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,
6972– 6976
52. Schenborn, E. T., and Mirendorf, R. C., Jr. (1985) Nucleic Acids Res. 13,
6223– 6236
53. Triana-Alonso, F. J., Dabrowski, M., Wadzack, J., and Nierhaus, K. H. (1995)
J. Biol. Chem. 270, 6298 – 6307
54. Martin, C. T., Muller, D. K., and Coleman, J. E. (1988) Biochemistry 27,
3966 –3974
55. Sastry, S., and Hoffman, P. L. (1995) Biochem. Biophys. Res. Commun. 211,
106 –114
56. Shi, Y., Gamper, H., and Hearst, J. E. (1988) J. Biol. Chem. 263, 527–534
57. Smeekens, S. P., and Romano, L. J. (1986) Nucleic Acids Res. 14, 2811–2827
58. Chamberlin, M. J., and Ring, J. (1973) J. Biol. Chem. 248, 2245–2250
59. Maitra, U., and Hurwitz, J. (1967) J. Biol. Chem. 242, 4897– 4907
60. Krummel, B., and Chamberlin, M. J. (1989) Biochemistry 28, 7829 –7842
61. Wang, D., and Hawley, D. K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 843– 847
62. Bonner, G., Lafer, E. M., and Sousa, R. (1994) J. Biol. Chem. 269,
25129 –25136
63. Zhou, W., Reines, D., and Doesctch, P. W. (1995) Cell 82, 577–585
64. Izban, M. G., Samkurashvili, I., and Luse, D. S. (1995) J. Biol. Chem. 270,
2290 –2297
65. Osterman, H. L., and Coleman, J. E. (1981) Biochemistry 20, 4884 – 4892
66. Krupp, G. (1989) Nucleic Acids Res. 17, 3023–3036
67. Markovtsov, V., Mustaev, A., and Goldfarb, A. (1996) Proc. Natl. Acad. Sci.
U. S. A. 93, 3221–3226
68. Hausman, R. (1988) in The Bacteriophages (Calendar, R., ed) pp. 259 –288,
Plenum Publishing Corp., New York
69. Nakai, H., and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208 –15216
70. Sadowski, P. (1985) in Nucleases (Linn, S. M., and Roberts, R. J., eds) pp.
23– 40, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
71. Sastry, S. S., and Ross, B. M. (1997) Biochemistry, in press
72. Gotte, M., Fackler, S., Hermann, T., Perola, E., Cellai, L., Gross, H. J., and
Heumann, H. EMBO J. 14, 833– 841
73. Richetti, M., and Buc, H. (1996) Biochemistry 35, 14970 –14983